WO2000021362A1 - VIABLE PrP (PRION PROTEIN) TRANSGENIC ANIMALS AND METHODS OF USE - Google Patents

Abstract

The present invention comprises non-human transgenic animals with: 1) a genome artificially altered to decrease or ablate endogenous PrP activity; and 2) proper health and development maintained through the administration of copper. The transgenic animal may have an endogenous gene altered to decrease endogenous PrP expression; ablated to eliminate endogenous PrP expression; or modified to express an exogenous form of PrP. The exogenous form of PrP includes a PrP gene from a genetically diverse species, a chimeric PrP gene containing sequences of the endogenous PrP gene from the PrP gene of a genetically diverse species; a mutated, deleted or otherwise altered form of the endogenous PrP, and PrP genes operably linked to an inducible promoter. Transgenic mouse of the invention are useful bioassay with their sensitivity to prion infection enhanced via the administration of controlled amounts of copper. Transgenic farm animals (e.g., cows) are useful for normal purposes with the benefit of not being susceptible to prions that normally infect animals of that species (e.g., BoPrPSc).

Description

VIABLE PrP (PRION PROTEIN) TRANSGENIC ANIMALS AND METHODS OF USE

GOVERNMENT RIGHTS The United States Government may have certain rights in this application pursuant to

Grant No. AG02132, AG10770, NS22786, NS14069, and NS07219 awarded by the National Institutes of Health.

FIELD OF THE INVENTION The invention relates generally to the field of non-human, transgenic animals altered with respect to the expression of the PrP gene.

BACKGROUND OF THE INVENTION Prions are infectious pathogens that cause central nervous system spongiform encephalopathies in animals. Prions are distinct from bacteria, viruses and viroids. The predominant hypothesis at present is that no nucleic acid component is necessary for infectivity of prion protein. Further, a prion which infects one species of animal (e.g., a human) will not infect another (e.g., a mouse).

The prion protein (PrP) was discovered by proressively enriching fractions from rodent brain for scrapie infectivity (Prusiner et al., 1982). Subsequently, the infectious, disease-causing PrP isoform was designated PrP^Sc molecules. At present, it appears that the scrapie isoform of the prion protein (PrP^Sc) is necessary for both the transmission and pathogenesis of the transmissible neurodegenerative diseases of animals and humans. See Prusiner, S.B., "Molecular biology of prion disease," Science 252:1515-1522 (1991). The most common prion diseases of animals are scrapie of sheep and goats and bovine spongiform encephalopathy (BSE) of cattle [Wilesmith, J. and Wells, Microbiol. Immunol. 772:21-38 (1991)]. Four prion diseases of humans have been identified: (1) kuru, (2) Creutzfeldt- Jakob Disease (CJD), (3) Gerstmann-Strassler-Scheinker Disease (GSS), and (4) fatal familial insomnia (FFI) [Gajdusek, D.C., Science 797:943-960 (1977); Medori et al., N. Engl. J. Med. 52f5:444-449 (1992)].

The presentation of human prion diseases as sporadic, genetic and infectious illnesses initially posed a conundrum which has been explained by the cellular genetic origin of PrP. The discovery of PrP^c occurred in response to the unexpected observation that the level of PrP mRNA in normal, control animals was the same as that in ill, scrapie, infected rodents (Chesebro et al, 1985, Oesch et al., 1985). PrP^c was readily distinguished from PrP^Sc because is was hydro lyzed under conditions that produced PrP 27-30 from PrP^Sc and PrP^c could be solubilized by non-denaturing detergents while PrP^Sc remained insoluble (Meyer et al, 1986).

The discovery of PrP^c under the circumstances where a disease process was being investigated gave no clue as to the function of this highly conserved protein. Though the discovery that PrP^c carries a glycosylphosphatidyl inositol (GPI) moiety provided insight into the subcellular trafficking of this protein, it did not reveal the function of PrP (Stahl et al, 1987; Taraboulos et al., 1995). The high histidine content of PrP raised the possibility the immobilized metal ion affinity chromatography (IMAC) might be employed in its purification (Sulkowski, 1989). IMAC with Cu²⁺ proved efficacious in the purification of PrP^c under non-denaturing conditions and this led to the finding that of PrP^c and PrP^Sc adopt dramatically different secondary and tertiary structures (Pan et al., 1993; Pan et al., 1992). Subsequently, the levels of Cu²⁺ were shown to be reduced -80% in microsomal membranes isolated from the brains of PrP deficient (Prnp⁰⁰) mice (Brown et al., 1977a) but despite this reduction in Cu²⁺ levels, the Prnp^{0 0} mice initially displayed no phenotype (Bϋehler et al., 1992). Mice with an ablated endogenous PrP gene were also found to not be susceptible to prion-mediated disorders due to the absence of the functional endogenous PrP protein. See U.S. Pat No. 5,698,763. Although these animals appeared to develop and survive like their normal counterparts, it has been found that these mice display certain dietary sensitivities that were not discovered in the initial studies of the Prnp^0/0 animals. Upon specific dietary deprivation, the Prnp^0/0 animals exhibited severe and debilitating pathological phenotypes which were not seen in similarly deprived normal mice.

There is a need in the art to provide animals with reduced or absent PrP expression that are immune to prion-mediated disorders normally affecting animals of that species and yet develop and survive without pathological phenotypes associated with the lowered endogenous PrP expression. SUMMARY OF THE INVENTION Animals with reduced expression of endogenous PrP deprived of dietary Cu²⁺ develop a number of pathologies including tremor and ataxia progressing to death. These animals also display reduced ceruloplasmin activity in serum and widespread astrocytic gliosis in their brain stem and cerebellum. The present invention comprises non-human transgenic animals with 1 ) a genome artificially altered to decrease or ablate endogenous PrP activity and 2) proper health and development maintained through the administration of copper. The transgenic animal may have an endogenous gene: altered to decrease endogenous PrP expression; ablated to eliminate endogenous PrP expression; or modified to express an exogenous form of PrP. The exogenous form of PrP includes a PrP gene from a genetically diverse species, a chimeric PrP gene containing sequences of the endogenous PrP gene from the PrP gene of a genetically diverse species; a mutated, deleted or otherwise altered form of the endogenous PrP, and PrP genes operably linked to an inducible promoter. Preferably, the transgenic animal of the invention is a rat, a hamster, or more preferably a mouse.

One object of the invention is to provide a transgenic, non-human animal which has its endogenous PrP gene altered to decrease or eliminate PrP expression. The pathological phenotype displayed by these animals upon copper deprivation is suppressed by the administration and/or monitoring of copper to the animals, which suppresses the pathological phenotype associated with reduced PrP expression. Copper is preferably maintained through the dietary administration of Cu2+ and/or monitoring of dietary Cu2+ levels. Preferably, the animals of this invention have an ablated endogenous PrP gene, and the transgenic animal is avian or mammalian and is provided a diet or is administered a formulation which comprises Cu2+ or provides Cu2+ to the animal upon digestion and/or administration. The diet or formulation of Cu2+ is provided in an amount sufficient to suppress pathologies of mammals not provided with such.

One advantage of the invention is that it provides animals resistant to prion infection that remain healthy and develop normally.

Another object of the invention is to provide a transgenic, non-human animal which has its genome altered to 1) decrease endogenous PrP expression and 2)express an exogenous PrP gene. Preferably, the transgenic animal has an ablated endogenous PrP gene The exogenous gene may be from a genetically diverse animal or a manipulated PrP gene such as a chimeric PrP gene comprised of codons from the host mammal and a genetically diverse mammal. Alternatively, the exogenous gene may be from genetically similar animals.

The pathological phenotype displayed by these animals upon copper deprivation is suppressed by the administration and/or monitoring of copper to the animals, which suppresses the pathological phenotype associated with reduced PrP expression.

One advantage of the invention is that it provides PrP ablated animals that can be maintained by copper and that are susceptible to prions that normally infect genetically diverse species.

A feature of the invention is that the PrP gene of the transgenic animal can be altered by replacing codons with codons of a test animal at the same relative position which differ from the codons of the host animal, up to and including replacing all the differing codons wherein the codons are replaced in a manner so as to maintain the operability of the gene. Yet another object of the invention is to provide a transgenic, non-human animal which has its genome altered to 1) decrease endogenous PrP expression and 2)express an exogenous PrP gene with an inducer sequence which affects the expression of the exogenous

PrP gene. Preferably, the transgenic animal has an ablated endogenous PrP gene. The pathological phenotype displayed by these animals upon copper deprivation is suppressed by the administration and/or monitoring of copper to the animals, which suppresses the pathological phenotype associated with reduced PrP expression. Yet another object of the invention is to provide for a method of testing samples for the presence of prions. The method involves providing transgenic animals with ablated endogenous PrP genes, maintaining said animals by the administration of copper, and inoculating the animal with material suspected of containing prions.

An advantage of the present invention is that the transgenic animal can be used to assay for the presence of prions in a genetically diverse sample without an intervening pathological phenotype caused by decreased endogenous PrP expression.

Another advantage is that transgenic animals inoculated with prions of humans can be used as test animals for testing drugs for efficacy in the treatment of humans suffering from diseases resulting from infection with prions. Another advantage is that the transgenic and hybrid animals can detect prions in a sample at very low levels, e.g., 1 part per million, and even as low as 1 part per billion. Still another advantage is that the transgenic and hybrid animals provide an assay which is highly accurate, i.e., does not provide false positives and consistently determines the presence of prions.

These and other objects, advantages, and eatures of the invention will become apparent to those persons skilled in the art upon reading the details of the chimeric gene, assay method, and transgenic mouse as more fully described below.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a bar graph illustrating ceruloplasmin levels in copper deprived mice. Ceruloplasmin activity is measured in the sera of PrnP⁰⁰, FVB and Tg(MoPrP-A)4053/FVB mice, both on a normal and on a copper-deprived diet.

Figure 2 is a survival curve of neonatal mice maintained on a copper restricted diet. Percent survival is plotted as a function of the time neonatal mice were fed a copper deficient diet. PrnP^0/0, FVB and Tg(MoPrP-A)4053/FVB neonatal pups were copper deprived beginning at four days of age.

Figure 3 is a bar graph showing a semi-quantitative assessment of reactive astrocytic gliosis in the cerebellar cortex of mice fed a normal diet (+Cu) and mice on a copper deficient diet (-Cu). KO=PrnP⁰⁰, WT=FVB, and Tg=Tg(MoPrP-A)4053/FVB.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Before the present proteins, assay methodology, and transgenic animals used in the assay are described, it is to be understood that this invention is not limited to particular protein, assay methods, chimeric and artificial genes, prion preparation or transgenic and hybrid animals described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

The publications discussed herein are provided solely for their disclosure prior to the fling date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

In reading this disclosure, it is pointed out that significant positions described the invention as it relates specifically to PrP genes, prions and prion diseases. However, those skilled in the art will understand that the invention is broader than such and includes transgenic animals containing a wider range of gene which genes are often lethal (50% or more, likely 75 to 95% or more fatalities) if expressed during early development.

DEFINITIONS The terms "treatment", "treating" and "treat" and the like are used herein to generally mean obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease (e.g., a prion disease) or symptom thereof and/or may be therapeutic in terms of partially or completely curing a disease (e.g., a prion disease) or adverse effect attributable to the disease. The "treatment" as used herein covers any treatment of a disease in a mammal, particularly a cow, pig, sheep, mouse or human, and includes:

(a) preventing a disease such as a prion disease or symptoms from occurring in a subject which may be predisposed to the disease or symptom or infected with a material such as prion particles but has not yet been diagnosed as having a disease which can include the use of gene therapy;

(b) inhibiting disease symptoms, i.e., arresting the development of a disease such as prion disease; or

(c) relieving a disease such as prion disease symptom, i.e., causing regression of a disease, e.g., a prion disease or prion disease symptoms. The term "gene detrimental to early development" refers to an exogenous gene which if expressed during early development (from conception to adulthood) would hinder development of the transgenic animal. The invention is most useful if the exogenous gene is lethal to the animal if expressed during early development or is at least lethal to a majority of the offspring, causing 50% or more, 75% or more, 95% or more or even 100% lethality. With the invention, the gene is turned off during early development and on during adulthood. Preferred genes detrimental to early development are those with an adverse effect on the nervous system, e.g., a PrP gene or α-synuclein gene.

The term "isolated" shall mean separated away from its natural environment. An isolated protein is not necessarily separated away from all materials it is normally present with and may remain glycosylated.

The term "FVB" refers to a mouse strain commonly used in the production of transgenic mice. For purposes of this invention it should be noted that the mouse prion protein (PrP) gene is intact and mouse PrP is therefore expressed at normal levels.

The term "Prnp-^0,0 or Prnp-Abl" refers to a transgenic animal which has its PrP gene ablated with the "^0/0" indicating that both alleles are ablated whereas ^o/+ indicates only one is ablated. Specifically, the animal being referred to is generally a transgenic mouse which has its PrP gene ablated i.e., a PrP knockout mouse. In that the PrP gene is disrupted no mouse PrP protein is expressed.

The term "sporadic CJD" abbreviated as "sCJD" refers to the most common manifestation of Creutzfeldt- Jakob Disease (CJD). This disease occurs spontaneously in individuals with a mean age of approximately 60 at a rate of 1 per million individuals across the earth.

The term "Iatrogenic CJD" abbreviated as "iCJD" refers to disease resulting from accidental infection of people with human prions. The most noted example of such is the accidental infection of children with human prions from contaminated preparations of human growth hormone. The term "Familial CJD" refers to a form of CJD which occurs rarely in families and is inevitably caused by mutations of the human prion protein gene. The disease results from an autosomal dominant disorder. Family members who inherit the mutations succumb to CJD.

The term "Gerstmann-Strassler-Scheinker Disease" abbreviated as "GSS" refers to a form of inherited human prion disease. The disease occurs from an autosomal dominant disorder. Family members who inherit the mutant gene succumb to GSS. The term "prion" shall mean an infectious particle known to cause diseases (spongiform encephalopathies) in humans and animals. The term "prion" is a contraction of the words "protein" and "infection" and the particles are comprised largely if not exclusively of PrP^Sc molecules encoded by a PrP gene which expresses PrP^c which changes conformation to become PrP^Sc. Prions are distinct from bacteria, viruses and viroids. Known prions include those which infect animals to cause scrapie, a transmissible, degenerative disease of the nervous system of sheep and goats as well as bovine spongiform encephalopathies (BSE) or mad cow disease and feline spongiform encephalopathies of cats. Four prion diseases known to affect humans are (1) kuru, (2) Creutzfeldt- Jakob Disease (CJD), (3) Gerstmann-Strassler-Scheinker Disease (GSS), and (4) fatal familial insomnia (FFI). As used herein prion includes all forms of prions causing all or any of these diseases or others in any animals used ~ and in particular in humans and in domesticated farm animals.

The terms "PrP gene" and "prion protein gene" are used interchangeably herein to describe genetic material which expresses proteins as shown in Figures 3-5 and polymorphisms and mutations such as those listed herein under the subheading "Pathogenic Mutations and Polymorphisms." Unless stated otherwise the term refers to the native wild- type gene and not to an artificially altered gene. The PrP gene can be from any animal including the "host" and "test" animals described herein and any and all polymorphisms and mutations thereof, it being recognized that the terms include other such PrP genes that are yet to be discovered. The term "PrP gene" refers generally to any gene of any species which encodes any form of a PrP amino acid sequences including any prion protein. Some commonly known PrP sequences are described in Gabriel et al., Proc. Natl. Acad. Sci. USA 59:9097-9101 (1992) which is incorporated herein by reference to disclose and describe such sequences.

The terms "standardized prion preparation," "prion preparation," "preparation" and the like are used interchangeably herein to describe a composition containing prions which composition is obtained from brain tissue of mammals which contain substantially the same genetic material as relates to PrP proteins, e.g., brain tissue from a set of mammals which exhibit signs of prion disease which mammals may comprise any of (1) a PrP chimeric transgene; (2) have an ablated endogenous PrP gene; (3) have a high copy number of PrP genes from a genetically diverse species; or (4) are hybrids with an ablated endogenous PrP gene and a PrP gene from a genetically diverse species. The mammals from which standardized prion preparations are obtained exhibit clinical signs of CNS dysfunction as a result of inoculation with prions and/or due to developing the disease due to their genetically modified make up, e.g., high copy number of PrP genes. The term "chimeric PrP gene" describes recombinantly constructed genes which when included in the genome of a host animal (e.g., a mouse) will render the mammal susceptible to infection from prions which naturally only infect a genetically diverse test mammal, e.g., human, bovine or ovine. In general, an artificial gene will include the codon sequence of the PrP gene of the mammal being genetically altered with one or more (but not all, and generally less than 40) codons of the natural sequence being replaced with a different codon — preferably a corresponding codon of a genetically diverse mammal (such as a human). The genetically altered mammal being used to assay samples for prions which only infect the genetically diverse mammal. A chimeric gene will, when inserted into the genome of a mammal of the host species, render the mammal susceptible to infection with prions which normally infect only mammals of the second species. A useful chimeric gene is MHu2M which contains the starting and terminating sequence of a mouse PrP gene and a nonterminal sequence region which is replaced with a corresponding human sequence which differs from a mouse PrP gene in a manner such that the protein expressed thereby differs at nine residues. Examples of such chimeric genes can be found in U.S. Patent No. 5,565,186 which is herein incorporated by reference.

The terms "host animal" and "host mammal" are used to describe animals which will have their genome genetically and artificially manipulated so as to include genetic material which is not naturally present within the animal. For example, host animals include mice, hamsters and rats which have their endogenous PrP gene altered by the insertion of an artificial gene or by the insertion of a native PrP gene of a genetically diverse test animal. The terms "test animal" and "test mammal" are used to describe the animal which is genetically diverse from the host animal in terms of differences between the PrP gene of the host animal and the PrP gene of the test animal. The test animal may be any animal for which one wishes to run an assay test to determine whether a given sample contains prions with which the test animal would generally be susceptible to infection. For example, the test animal may be a human, cow, sheep, pig, horse, cat, dog or chicken, and one may wish to determine whether a particular sample includes prions which would normally only infect the test animal. This is done by including PrP gene sequences of the test animal into the host animal, inoculating the host animal with prions which would normally only infect the test animal.

The terms "genetically diverse animal" and "genetically diverse mammal" are used to describe an animal which includes a native PrP codon sequence of the host animal which differs from the genetically diverse test animal by 17 or more codons, preferably 20 or more codons, and most preferably 28-40 codons. Thus, a mouse PrP gene is genetically diverse with respect to the PrP gene of a human, cow or sheep, but is not genetically diverse with respect to the PrP gene of a hamster. In general, prions of a given animal will not infect a genetically diverse animal.

The terms "ablated prion protein gene," "disrupted PrP gene," "ablated PrP gene," "PrP^%" and the like are used interchangeably herein to mean an endogenous prion protein gene which has been altered (e.g., add and/or remove nucleotides) in a manner so as to render the gene inoperative. Examples of nonfunctional PrP genes and methods of making such are disclosed in Bϋeler, H., et al "Normal development of mice lacking the neuronal cell-surface PrP protein" Nature 356, 577-582 (1992) which is incorporated herein by reference. Both alleles of the genes are disrupted.

The terms "hybrid animal," "transgenic hybrid animal" and the like are used interchangeably herein to mean an animal obtained from the cross-breeding of a first animal having an ablated endogenous PrP gene with a second animal which includes either (1) a chimeric gene or artificial PrP gene or (2) a PrP gene from a genetically diverse animal. For example a hybrid mouse is obtained by cross-breeding a mouse with an ablated mouse PrP gene with a mouse containing (1) human PrP genes (which may be present in high copy numbers) or (2) chimeric genes. The term hybrid includes any offspring of a hybrid including inbred offspring of two hybrids provided the resulting offspring is susceptible to infection with prions with normal infect only a genetically diverse species.

The terms "susceptible to infection" and "susceptible to infection by prions" and the like are used interchangeably herein to describe a transgenic or hybrid test animal which develops a prion disease if inoculated with prions which would normally only infect a genetically diverse test animal. The terms are used to describe a transgenic or hybrid animal such as a transgenic mouse Tg(MHu2M) which, without the chimeric PrP gene, would not be susceptible to infection with a human prion (less than 20% chance of infection) but with the chimeric gene is susceptible to infection with human prions (80% to 100% chance of infection).

The terms "resistant to infection", "resistant to infection with prions" and the like mean the animal includes a PrP gene which renders the animal resistant to prion disease when inoculated with an amount and type of prion which would be expected to cause prion disease in the animal.

The term "incubation time" shall mean the time from inoculation of an animal with a prion until the time when the animal first develops detectable symptoms of disease resulting from the infection. A reduced incubation time is six months or less, preferably about 75 days ± 25 days or less, more preferably about 30 days ± 10 days or less.

The term "suppress" shall mean to lessen the effects of a visible, undesirable phenotype. Although suppression may not be an complete elimination of the phenotype, it preferably alleviates the phenotype to the extent that the phenotype will not interfere with normal development, health, and/or behavior of the animal. In a preferred embodiment, suppression of a pathological phenotype results in mice in which the phenotype is undetectable.

ABBREVIATIONS USED HEREIN INCLUDE:

BSE for bovine spongiform encephalopathy; CJD for Creutzfeldt- Jakob Disease;

CNS for central nervous system;

FFI for fatal familial insomnia;

FVB for a standard inbred strain of mice often used in the production of transgenic mice since eggs of FVB mice are relatively large and tolerate microinjection of exogenous DNA relatively well;

GSS for Gerstmann-Strassler-Scheinker Disease;

Hu for human;

HuPrP for a human PrP protein;

Mhu2M for a chimeric mouse/human PrP gene wherein a region of the mouse PrP gene is replaced by a corresponding human sequence which differs from mouse PrP at 9 codons;

Mo for mouse; MoPrP for a mouse PrP protein;

MoPrP^Sc for the scrapie isoform of the mouse PrP protein; p_mp^θ/o f_{or a}bi_a ion of both alleles of an endogenous PrP protein gene, e.g., the Mo PrP gene; PrP^Sc for the scrapie isoform of the PrP protein;

ScN2a for persistently infected scrapie mouse neuroblastoma cells also expressing (MHM2)PrP^c;

SHa for a Syrian hamster; SHa PrP for a Syrian hamster PrP protein; Tg for transgenic;

Tg(BovPrP) for transgenic mice containing the complete cow PrP gene; Tg(HuPrP) for transgenic mice containing the complete human PrP gene; Tg(HuPrP)/Prnp^0/0 for a hybrid mouse obtained by crossing a mouse with a human PrP protein gene (HuPrP) with a mouse with both alleles of the endogenous PrP protein gene disrupted;

Tg(MHu2M) mice are transgenic mice of the invention which include the chimeric MHu2M gene;

Tg(MHu2M)/Prnp⁰⁰ for a hybrid mouse obtained by crossing a mouse with a chimeric PrP protein gene (MHu2M) with a mouse with both alleles of the endogenous PrP protein gene disrupted;

Tg(SHa PRP) for a transgenic mouse containing the PrP gene of a Syrian hamster; Tg(SHa PrP) for transgenic mice containing the complete sheep PrP gene. Tg(tetO-MoPrP) for transgenic mice containing an active portion of the tetracycline operon, the tetO-heptamer element, operably linked to the mouse PrP gene. Tg(tetO-LacZ/MoPrP) for transgenic mice containing a tetO-heptamer element, and both MoPrP-A and β-galactosidase (LacZ) on either side of the tetO heptamer in opposite orientation.

Tg(tetO-MoPrP/tTA) for double transgenic mice containing both the MoPrP gene operably linked to the tetO-heptamer element and a tetracycline transactivating element. Tg(tetO-MoPrP/rtTA) for double transgenic mice containing both the MoPrP gene operably linked to the tetO-heptamer element and a reverse tetracycline transactivating element. PrP NUCLEIC ACID COMPOSITIONS The term "PrP" is used generically to designate PrP genes, e.g. homologs from rat, human, mouse, guinea pig, etc. , and their alternate forms. Used generically, this term encompasses different isoforms, polymorphisms, variant sequences, and mutated forms of PrP as well. The term is also intended to mean the open reading frame encoding specific polypeptides, introns, and adjacent 5' and 3' non-coding nucleotide sequences involved in the regulation of expression, up to about 1 kb beyond the coding region, but possibly further in either direction. The DNA sequences encoding PrP may be cDNA or genomic DNA or a fragment thereof. The gene may be introduced into an appropriate vector for extrachromosomal maintenance or for integration into the host.

A genomic sequence of interest comprises the nucleic acid present between the initiation codon and the stop codon, as defined in the listed sequences, including all of the introns that are normally present in a native chromosome. It may further include the 3' and 5' untranslated regions found in the mature mRNA. It may further include specific transcriptional and translational regulatory sequences, such as promoters, enhancers, etc., including about 1 kb, but possibly more, of flanking genomic DNA at either the 5' or 3' end of the transcribed region. The genomic DNA may be isolated as a fragment of 100 kbp or smaller; and substantially free of flanking chromosomal sequence.

The sequence of this 5' region, and further 5' upstream sequences and 3' downstream sequences, may be utilized for promoter elements, including enhancer binding sites, that provide for expression in tissues where PrP is expressed. The tissue specific expression is useful for determining the pattern of expression, and for providing promoters that mimic the native pattern of expression. Naturally occurring polymorphisms in the promoter region are useful for determining natural variations in expression, particularly those that may be associated with disease. Alternatively, mutations may be introduced into the promoter region to determine the effect of altering expression in experimentally defined systems. Methods for the identification of specific DNA motifs involved in the binding of transcriptional factors are known in the art, e.g. sequence similarity to known binding motifs, gel retardation studies, etc. For examples, see Blackwell et al. (1995) Mol Med 1:194-205; Mortlock et al. (1996) Genome Res. 6:327-33; and Joulin and Richard-Foy (1995) Eur J Biochem 232:620-626. The regulatory sequences may be used to identify cis acting sequences required for transcriptional or translational regulation of PrP expression, especially in different tissues or stages of development, and to identify cis acting sequences and trans acting factors that regulate or mediate expression. Such transcription or translational control regions may be operably linked to a PrP gene in order to promote expression of wild type or altered PrP or other proteins of interest in cultured cells, or in embryonic, fetal or adult tissues, and for gene therapy.

The nucleic acid compositions used in the subject invention may encode all or a part of the PrP polypeptides as appropriate. Fragments may be obtained of the DNA sequence by chemically synthesizing oligonucleotides in accordance with conventional methods, by restriction enzyme digestion, by PCR amplification, etc. For the most part, DNA fragments will be of at least 15 nt, usually at least 18 nt, more usually at least about 50 nt. Such small DNA fragments are useful as primers for PCR, hybridization screening, etc. Larger DNA fragments, i.e. greater than 100 nt are useful for production of the encoded polypeptide. For use in amplification reactions, such as PCR, a pair of primers will be used.

Homologs of cloned PrP are identified by various methods known in the art. Nucleic acids having sequence similarity are detected by hybridization under low stringency conditions, for example, at 50°C and 10XSSC (0.9 M saline/0.09 M sodium citrate) and remain bound when subjected to washing at 55°C in lXSSC. Sequence identity may be determined by hybridization under stringent conditions, for example, at 50°C or higher and 0.1XSSC (9 mM saline/0.9 mM sodium citrate). By using probes, particularly labeled probes of DNA sequences, one can isolate homologous or related genes. The source of homologous genes may be any species, e.g. primate, rodents, canines, felines, bovines, ovines, equines, etc. The PrP sequence, including flanking promoter regions and coding regions, may be mutated in various ways known in the art to generate targeted changes in promoter strength, sequence of the encoded protein, etc. The sequence changes may be substitutions, insertions or deletions. Deletions may include large changes, such as deletions of a domain or exon. Other modifications of interest include epitope tagging, e.g. with the FLAG system, HA, etc. For studies of subcellular localization, fusion proteins with green fluorescent proteins (GFP) may be used. Such mutated genes may be used to study structure-function relationships of apo polypeptides, or to alter properties of the proteins that affect their function or regulation. Techniques for in vitro mutagenesis of cloned genes are known. Examples of protocols for scanning mutations may be found in Gustin et al, 1993 Biotechniques 14:22 ; Barany, 1985 Gene 37:111-23; Colicelli et al., 1985 Mol Gen Genet 199:537-9; and Prentki et al., 1984 Gene 29:303-13. Methods for site specific mutagenesis can be found in Sambrook et al., 1989 Molecular Cloning: A Laboratory Manual, CSH Press, pp. 15.3- 15.108; Weiner et al., 1993 Gene 126:35-41; Sayers et al., 1992 Biotechniques 13:592-6; Jones and Winistorfer, 1992 Biotechniques 12:528-30; Barton et al., 1990 Nucleic Acids Res 18:7349-55; Marotti and Tomich, 1989 Gene Anal Tech 6:67-70; and Zhu 1989 Anal Biochem 177:120-4. There are a number of mutations and polymorphisms existing with respect to the PrP gene of different species. A number of the mutations and polymorphisms are listed in the "Mutation Table" provided below. It is believed that additional mutations and polymorphisms exist in all species within the PrP gene. However, until now one could not determine which sequences might provide for resistance to prion disease. Animals with a PrP gene which is heterozygous at a particular point could be bred with other animals which are heterozygous at that point in order to produce offspring which include those with a homozygous PrP gene of the type desired. Substitutions in the PrP transgene may be made with an amino acid which is biochemically quite different from the amino acid at that position which is known to render the animal susceptible to prion infection. Thus, if a basic and/or polar amino acid is present at the critical site that site could be replaced with an acidic and/or nonpolar amino acid. With these criteria in mind some trial and error would be required. Acidic amino acids should be substituted with basic amino acids and vice versa. Polar amino acids should be substituted with nonpolar amino acids and vice versa. Such mutations may increase the susceptibility of the transgenic animals for the uses described herein.

There are a number of known pathogenic mutations in the human PrP gene. Further, there are known polymorphisms in the human, sheep and bovine PrP genes. The following is a list of such mutations and polymorphisms: MUTATION TABLE

Pathogenic human Human Sheep Bovine mutations Polymorphisms Polymorphisms Polymorphisms

2 octarepeat insert Codon 129 Met/Val Codon 171 Arg/Gln 5 or 6 octarepeats 4 octarepeat insert Codon 219 Glu/Lys Codon 136 Ala/Nal

5 octarepeat insert

6 octarepeat insert

7 octarepeat insert

8 octarepeat insert 9 octarepeat insert

Codon 102 Pro-Leu

Codon 105 Pro-Leu

Codon 117 Ala-Val

Codon 145 Stop Codon 178 Asp-Asn

Codon 180 Val-Ile

Codon 198 Phe-Ser

Codon 200 Glu-Lys

Codon 210 Val-Ile Codon 217 Asn-Arg

Codon 232 Met-Ala

In order to provide further meaning to the above chart demonstrating the mutations and polymorphisms, one can refer to the published sequences of PrP genes. For example, a chicken, bovine, sheep, rat and mouse PrP gene are disclosed and published within Gabriel et al., Proc. Natl. Acad. Sci. USA 59:9097-9101 (1992). The sequence for the Syrian hamster is published in Basler et al., Cell 46:411-428 (1986). The PrP gene of sheep is published by Goldmann et al., Proc. Natl. Acad. Sci. USA 57:2476-2480 (1990). The PrP gene sequence for bovine is published in Goldmann et al, J. Gen. Virol. 72:201-204 (1991). The sequence for chicken PrP gene is published in Harris et al., Proc. Natl. Acad. Sci. USA 55:7664-7668 (1991). The PrP gene sequence for mink is published in Kretzschmar et al., Gen. Virol. 75:2757-2761 (1992). The human PrP gene sequence is published in Kretzschmar et al, DNA 5:315-324 (1986). The PrP gene sequence for mouse is published in Locht et al, Proc. Natl. Acad. Sci. USA 55:6372-6376 (1986). The PrP gene sequence for sheep is published in Westaway et al., Genes Dev. 5:959-969 (1994). These publications are all incorporated herein by reference to disclose and describe the PrP gene and PrP amino acid sequences.

Certain PrP deletion constructs have been shown to produce smaller, more soluble PrP protein that are still capable of producing the scrapie form of the protein. These constructs may be incorporated into transgenes with altered expression. One example of such a transgene is mouse PrP 106, which lacks residues 23-88 and 141-176. and has a six histidine tag in-frame bridging residues 140 and 177, is converted into a PrP^Sc like molecule. Both full-length and fragment transgenes may also include non-PrP sequences, such as epitope tags.

TRANSGENIC ANIMALS The term "transgene" is used herein to describe genetic material that has been or is about to be artificially inserted into the genome of a cell, particularly a mammalian cell for implantation into a living animal. The transgene is used to transform a cell, meaning that a permanent or transient genetic change, preferably a permanent genetic change, is induced in a cell following incorporation of exogenous DNA. A permanent genetic change is generally achieved by introduction of the DNA into the genome of the cell. Vectors for stable integration include plasmids, retroviruses and other animal viruses, YACs, and the like. Of interest are transgenic mammals, e.g. cows, pigs, goats, horses, etc., and particularly rodents, e.g. rats, mice, etc.

Transgenic animals comprise an exogenous nucleic acid sequence present as an extrachromosomal element or stably integrated in all or a portion of its cells, especially in germ cells. Unless otherwise indicated, it will be assumed that a transgenic animal comprises stable changes to the germline sequence. During the initial construction of the animal, "chimeras" or "chimeric animals" are generated, in which only a subset of cells have the altered genome. Chimeras are primarily used for breeding purposes in order to generate the desired transgenic animal. Animals having a heterozygous alteration are generated by breeding of chimeras. Male and female heterozygotes are typically bred to generate homozygous animals. Transgenic animals fall into two groups, colloquially termed "knockouts" and

"knockins". In the present invention, knockouts have a partial or complete loss of function in one or both alleles of the endogenous PrP gene. Knockins have an introduced transgene with altered genetic sequence and function from the endogenous gene. The two may be combined, such that the naturally occurring gene is disabled, and an altered form introduced.

In a knockout, preferably the target gene expression is undetectable or insignificant. A knock-out of a PrP gene means that function of the PrP receptor has been substantially decreased so that expression is not detectable or only present at insignificant levels. In the present invention, transgenic knockouts have a partial or complete loss of function in one or both alleles of the endogenous PrP gene. This may be achieved by a variety of mechanisms, the preferred being homologous recombination. See e.g. U.S. Patents 5,464,764, 5,627,059 and related patents and publications to Capecchi et al., which are incorporated herein in their entirety. Other mechanisms include introduction of a disruption of the coding sequence, e.g. insertion of one or more stop codons, insertion of a DNA fragment, etc. , deletion of coding sequence, substitution of stop codons for coding sequence, etc. In some cases the exogenous transgene sequences are ultimately deleted from the genome, leaving a net change to the native sequence. Different approaches may be used to achieve the "knock-out". A chromosomal deletion of all or part of the native gene may be induced, including deletions of the non-coding regions, particularly the promoter region, 3' regulatory sequences, enhancers, or deletions of gene that activate expression of PrP genes. A functional knock-out may also be achieved by the introduction of an anti-sense construct that blocks expression of the native genes (for example, see Li and Cohen (1996) Cell 85:319-329). "Knock-outs" also include conditional knock-outs, for example where alteration of the target gene occurs upon exposure of the animal to a substance that promotes target gene alteration, introduction of an enzyme that promotes recombination at the target gene site (e.g. Cre in the Cre-lox system), or other method for directing the target gene alteration postnatally.

A "knock-in" of a target gene means an alteration in a host cell genome that results in altered expression or function of the native PrP gene. Increased (including ectopic) or decreased expression may be achieved by introduction of an additional copy of the target gene, or by operatively inserting a regulatory sequence that provides for enhanced expression of an endogenous copy of the target gene. These changes may be constitutive or conditional, i.e. dependent on the presence of an activator or represser. The exogenous gene is usually either from a different species than the animal host, or is otherwise altered in its coding or non-coding sequence. The introduced gene may be a wild-type gene, naturally occurring polymorphism, or a genetically manipulated sequence, for example having deletions, substitutions or insertions in the coding or non-coding regions. The introduced sequence may encode a PrP polypeptide, or may utilize the PrP promoter operably linked to a reporter gene. Where the introduced gene is a coding sequence, it is usually operably linked to a promoter, which may be constitutive or inducible, and other regulatory sequences required for expression in the host animal. By "operably linked" is meant that a DNA sequence and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules, e.g. transcriptional activator proteins, are bound to the regulatory sequence(s).

Specific constructs of interest, but are not limited to, include anti-sense PrP, which will block native PrP expression, expression of dominant negative PrP mutations, and over-expression of a PrP gene. A detectable marker, such as lac Zmay be introduced into the locus, where upregulation of expression will result in an easily detected change in phenotype. Constructs utilizing the PrP promoter region, in combination with a reporter gene or with the coding region are also of interest. A series of small deletions and/or substitutions may be made in the PrP gene to determine the role of different exons in DNA binding, transcriptional regulation, etc. By providing expression of PrP protein in cells in which it is otherwise not normally produced, one can induce changes in cell behavior.

The exogenous PrP gene may be any mammalian PrP gene, and may be a wild-type gene, a naturally occurring polymorphism, or a genetically manipulated sequence, for example having deletions, substitutions or insertions in the coding or non-coding regions. The introduced sequence may encode a PrP polypeptide, or may utilize the PrP promoter operably linked to a reporter gene. Where the introduced gene is a coding sequence, it is usually operably linked to a promoter, which may be constitutive or inducible, and other regulatory sequences required for expression in the host animal. By "operably linked" is meant that a DNA sequence and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules, e.g. transcriptional activator proteins, are bound to the regulatory sequence(s).

Specific constructs of interest include, but are not limited to, anti-sense PrP, which will block native PrP expression, expression of dominant negative PrP mutations, and over-expression of a PrP gene. A detectable marker, such as lac Z may be introduced into the locus, where upregulation of expression will result in an easily detected change in phenotype. Constructs utilizing the PrP promoter region, in combination with a reporter gene or with the coding region are also of interest.

DNA constructs for homologous recombination will comprise at least a portion of the PrP gene with the desired genetic modification, and will include regions of homology to the target locus. DNA constructs for random integration need not include regions of homology to mediate recombination. Conveniently, markers for positive and negative selection are included. Methods for generating cells having targeted gene modifications through homologous recombination are known in the art. For various techniques for transfecting mammalian cells, see Keown et al. (1990) Methods in Enzymology 185:527-537. For embryonic stem (ES) cells, an ES cell line may be employed, or embryonic cells may be obtained freshly from a host, e.g. mouse, rat, guinea pig, etc. Such cells are grown on an appropriate fibroblast-feeder layer or grown in the presence of appropriate growth factors, such as leukemia inhibiting factor (LIF). When ES cells have been transformed, they may be used to produce transgenic animals. See U.S. Patents 5,387,742, 4,736,866 and 5,565,186 for methods of making transgenic animals. After transformation, the cells are plated onto a feeder layer in an appropriate medium. Cells containing the construct may be detected by employing a selective medium. After sufficient time for colonies to grow, they are picked and analyzed for the occurrence of homologous recombination or integration of the construct. Those colonies that are positive may then be used for embryo manipulation and blastocyst injection. Blastocysts are obtained from 4 to 6 week old superovulated females. The ES cells are trypsinized, and the modified cells are injected into the blastocoel of the blastocyst. After injection, the blastocysts are returned to each uterine horn of pseudopregnant females. Females are then allowed to go to term and the resulting litters screened for mutant cells having the construct. By providing for a different phenotype of the blastocyst and the ES cells, chimeric progeny can be readily detected.

The chimeric animals are screened for the presence of the modified gene and males and females having the modification are mated to produce homozygous progeny. If the gene alterations cause lethality at some point in development, tissues or organs can be maintained as allergenic or congenic grafts or transplants, or in in vitro culture. A number of different transgenic PrP animals can be made in the practice of the invention. The production and use of certain PrP transgenic animals are described in USSN 08/692,892 and 09/052,963 which are incorporated herein by reference. Crossed With MoPrP Gene Ablated Mice

FVB mice expressing human PrP genes have been constructed using the cos.SHaTet cosmid expression vector derived from the Syrian hamster (SHa). The FVB strain of mice contain and express the normal complement of MoPrP genes and so one method for introducing the HuPrP transgene array into a background in which MoPrP expression is ablated is by genetic crosses between the transgenic FVB-derived line and a second line of transgenic mice in which both MoPrP genes were disrupted. Mice homozygous for the disrupted Prnp genes were created. These genetically-altered mice were created by a process known as homologous recombination (Thomas and Capecchi, Cell 51:503-512, 1987) in which a selectable disrupted MoPrP gene was introduced into embryonic stem (ES) cells from SV129 mice. Blastocysts of C57BL/6J mice were injected with SV129 ES cells in which one copy of the MoPrP gene had been disrupted thus generating a chimeric mouse with one disrupted allele. That mouse was mated with a C57BL mouse and the offspring crossed to each other to produce null animals in which both copies of the MoPrP gene were disrupted, referred to as Prnp^0/0 mice. Subsequently, these Prnp^0/0 mice were repeatedly crossed onto the FVB background. FVB-derived transgenic mouse lines Tg(HuPrP)FVB/152 and Tg(HuPrP)FVB/440 were crossed with Prnp^0/0 mice. Backcrossing these mice produced animals in which the only PrP^c molecules that were synthesized were those encoded by the transgene.

Producing Transgenic Mice Using Fertilized Oocytes From MoPrP Gene Ablated Mice

The second method for producing transgenic mice in which the only PrP^c molecules synthesized are encoded by the HuPrP transgene is by directly microinjecting DNA from a vector capable of directing expression of HuPrP. Derivatives of the cos.SHaTet cosmid expression vector containing the HuPrP open reading frame were used — (other expression systems could be used including a cosmid consisting of the cognate HuPrP gene or other vectors capable of appropriate expression of HuPrP in transgenic mice). Using embryos from the originally created C57BL-derived Prnp^0/0 mice we encountered great difficulty in producing transgenic mice by this method because of the poor survival rates of micro injected embryos. These Prnp⁰⁰ mice were subsequently repeatedly crossed onto the FVB background to produce mice which were genetically -95% FVB but which were also homozygous for the gene ablation. By modifying the Prnp^0/0 mice in this way we now have very high rates of production of transgenic mice by this method.

Gene Replacement A different approach to eliminating the inhibitory effects of MoPrP would be to create new lines of transgenic mice in which the endogenous MoPrP genes were replaced with HuPrP genes by homologous recombination in ES cells. This gene-replacement approach (Hasty et al., Nature 350:243-6, 1991 ; Valancius and Smithies, Mol. Cell Biol. 11 :1402-8, 1991) is a variation of the gene-insertion experiment described above in which Prnp^0/0 mice were created. In gene replacement, the sequences in the input DNA completely replace those in the target DNA. The methodologies that are currently available permit gene targeting at high efficiency and fidelity so that it should in theory be possible to replace the MoPrP gene with the homologous HuPrP gene in ES stem cells and thereby produce mice that are homozygous for this replacement. After completing gene replacement with either HuPrP of chimeric MHu2MPrP, these mice are mated to transgenic mice expressing high levels of the homologous protein such as HuPrP or MHu2MPrP. The mice will express the highest levels of the foreign PrP of interest and possess the shortest incubation times. For example, mice with -50 copies of the MoPrP transgene have incubation times of -60 days after inoculation with - 10⁶ ID₅₀ units if the endogenous MoPrP genes are ablated; in contrast, incubation times of -48 days were found if the endogenous MoPrP genes are left intact (Table 8). Alternatively, the fertilized eggs from these mice with gene replacements can be microinjected with the DNA encoding either the same PrP gene as that replaced or a related gene.

Animals with Inducible exogenous PrP genes

A different approach to eliminating the inhibitory effects of MoPrP would be to create new lines of transgenic mice in which the endogenous MoPrP genes were replaced with MoPrP genes or HuPrP genes operably linked to an inducible promoter. The expression of the exogenous PrP gene would be temporally and/or qualitatively using an inducible system such as that disclosed in Shockett, PNAS 43:5173-5176 (1996), which is incorporated by reference herein. The pioneering tet-regulated gene expression system involved a constitutive expression of the tet transactivator protein (tTA) with the cytomegalovirus (CMV) immediate early (IE) promoter/enhancer. A modified system has also been developed using a reverse transactivator (rtTA) that binds tetO efficiently only in the presence of the tet derivatives doxycycline or anhydrotetracycline. These and other similar systems may be used in the present invention without departing from the spirit of the disclosure, as will be obvious to those skilled in the art. For example, systems such as ecdysome inducible systems can be used instead of the tetracycline inducible system. The transactivator of the inducible system may be introduced via the CMV promoter, viral vectors driven by either the SV40 promoter, by glial-cell specific promoters, or by the autonomous parvo virus LuIII.

Transgenic Mice With Human and Chimeric PrP Genes

FVB mice expressing human, chimeric Hu/Mo and mutant PrP genes were constructed using the cos.SHaTet cosmid expression vector derived from the Syrian hamster (SHa) (Scott et al., 1992). Table 1 below shows the designation of the mouse line, the expressed PrP^c molecules and the approximate level of transgenic expression. Also indicated are those mouse lines that were crossed with Prnp^0/0 mice in which the mouse PrP gene had been disrupted by homologous recombination (Bϋeler et al., 1992). Backcrossing these mice produced animals in those encoded by the transgene. While SV129ES cells were used to generate a chimeric mouse with a disrupted PrP allele, that mouse was mated with a C57BL mouse and the offspring crossed to each other to produce null animals.

Subsequently, these Prnp⁰⁰ mice were repeatedly crossed onto the FVB background.

Nomenclature and Characateristics of Transgenic Mouse Lines

Mouse Line Description Expressed PrP Transgene Sequence⁰

PrP^c Expression Molecules

(i) Tg(HuPrP) mice

Tg(HuPrP) Hu,Mo -4-8x V129

152/FVB

Tg(HuPrp) Hu -4-8x V129

152/Prbp^0/0 Tg(HuPrP) Hu -2x M129

440/Prnp⁰⁰

(ii) Tg(MHu2M) mice Tg(MHu2M) MHu2M, Mo ^■ lx M128

5378/FVB

Tg(MHu2M) MHu2M ^•lx M128

5378/ _Pmpo/o Tg(MHu2M- MHu2M-L -2x M128, L10

P101L)69/

Prnn"

COPPER ADMINISTRATION Copper may be administered to the animals of the invention using any convenient means capable of administering the copper in the desired quantity. Thus, the agent can be incorporated into a variety of formulations for administration. More particularly, the agents of the present invention can be formulated into feed and/ or compositions by combination with appropriate materials. Formulations which contain copper and/or copper derivatives which result in copper which can be absorbed into a living organism can be injected or administered by any other means known to those skilled in the art. For administration via feed, i.e. dietary administration of the copper, the copper is added in a metabolically accessible form to the feed, for example as a chloride, sulfate and/or organic chelates, and the copper is ingested by the subject to be treated. Dietary ingestion of the copper is the method of administration of the preferred embodiment of the instant invention. For administration via pharmaceutical composition, pharmaceutically acceptable carriers or diluents may be added, and the formulation may be prepared in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols. As such, administration of the agents can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intratracheal, etc., administration.

For oral preparations, the agents can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxy methylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents. The agents can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives. The copper can be utilized in aerosol formulation to be administered via inhalation.

The compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifiuoromethane, propane, nitrogen and the like.

Furthermore, the copper can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. The copper used of the present invention can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more inhibitors. Similarly, unit dosage forms for injection or intravenous administration may comprise the inhibitor(s) in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.

Standardization prion preparations are produced for use in assays so as to improve the reliability of the assy. Preparation can be obtained from the animals of the invention and such preparations are produced as described in USSN 08/521,992 which is herein incorporated by reference.

TEST ANIMAL Although a variety of different test animals could be used for testing for the presence of prions within a sample, preferred host animals are mice and hamsters, with mice being most preferred in that there exists considerable knowledge on the production of transgenic animals. Other possible host animals include those belonging to a genus selected from Mus (e.g. mice), Rattus (e.g. rats), Oryctolagus (e.g. rabbits), and Mesocricetus (e.g. hamsters) and Cavia (e.g., guinea pigs). In general mammals with a normal full grown adult body weight of less than 1 kg which are easy to breed and maintain can be used. The host PrP gene can be changed to include codons from genetically diverse PrP genes from test animals belonging to a genus selected from Bos, Ovis, Sus and Homo. Preferably, a mouse host PrP gene is changed to include codons from a human, cow or sheep PrP gene, with human being most preferred. Humans are preferred because an important object of the invention is to use the animal to test a sample of material to determine if that material has prions which will infect a human and cause a human to develop a CNS disease such as CJD. Preferred transgenic animals are disclosed in U.S. Patent 5,565,186 issued October 15, 1996 and WO 97/04814 published February 13, 1997 which are incorporated herein by reference to disclose transgenic animals and methods of making and using such.

The genetic material which makes up the PrP gene is known for a number of different species of animals (see Gabriel et al., Proc. Natl. Acad. Sci. USA 59:9097-9101 (1992)). Further, there is considerable homology between the PrP genes in different mammals. For example, see the amino acid sequence of mouse PrP compared to human, cow and sheep PrP in Figures 3, 4 and 5 wherein only the differences are shown. Although there is considerable genetic homology with respect to PrP genes, the differences are significant in some instances. More specifically, due to small differences in the protein encoded by the PrP gene of different mammals, a prion which will infect one mammal (e.g. a human) will not normally infect a different mammal (e.g. a mouse). Due to this "species barrier," it is not generally possible to use normal animals, (i.e., animal which have not had their genetic material related to prions manipulated) such as mice to determine whether a particular sample contains prions which would normally infect a different species of animal such as a human.

EVALUATION OF EARLY LOCOMOTOR AND MOTOR COORDINATION DEFICITS FOR RAPID DIAGNOSIS OF CEREBELLAR SCRAPIE PROGRESSION The diagnosis of scrapie in rodents involves the detection of at least two classical neurological signs associated with prion diseases as well as the progression of these signs over time. Classical clinical signs are: agressivity, ataxia, dysmetria, tremor, head-bobbing, lack of righting reflex, convulsions, kyphosis, head tilt, tail rigidity, bradykinesia, proprioceptive deficits, masked facies, loss of deep pain sensation, circling and paralysis. The use of a system of the present invention, whether inducible or not, where PrP expression is predominantly localized within the cerebellum, facilitates the detection of replicating prions as the neurodegeneration can be followed by scoring ataxia or disorders of movement, gait, equilibrium and coordination associated with cerebellar degeneration.

To diagnose animals exhibiting scrapie sickness in a shorter period of time, a pressure-sensitive measurement system may be employed to detect and record an inoculated animal's physiological changes, and specifically its motor skills. One example of such a pressure sensitive system that would be effective in monitoring changes in walking is a platform with a piezoelectric ground reaction force (GRF) measurement system such as that used in the Gaitway Instrumented Treadmill (Kistler Biomechanics, Winterthut, Switzerland). Such a system has the ability to measure vertical ground reaction force and center of pressure for complete, multiple foot strikes. The system also has an integrated software system that can distinguish between left and right strikes and measure vertical force, center of pressure, and temporal gait parameters. A database can keep track of trials by subject name, identification number, or other user-specified classification. Multiple trials can be overlaid on a single graph, and the progression of a single subject examined over time. The time base can be varied to view data in absolute time, relative time, percent contact, percent step, and percent gait cycle.

Using such systems for the early detection of scrapie disease can also be achieved more by the systematic quantitative evaluation of these parameters over time using apparatus designed for such purpose.

Another method of evaluating motor coordination is a rotorod test (Sakaguchi et al.). Animals are placed on a rotating rod, whose rotating frequency is steadily increased from 10 rpm until the animals falls from it. The animal is subjected to ten trials and its best score recorded as its score on that given day. This test would offer a quantitative measurement of motor skills over the progression of prion disease and permit the early detection of motoric deficiencies.

Other systems with the ability to distinguish motor skill abilities may also be employed, as will be obvious to those skilled in the art.

DRUG EFFICACY EVALUATION Transgenic animals of the invention can be used to evaluate the efficacy of drugs. In its simplest form, the exogenous gene is turned on in two mice and the drug is administered to one but not the other mouse. The effect of the drug on inhibiting the progress of disease as compared to the mouse with no drug is then determined. In another method, the exogenous gene is turned on and left on until symptoms develop in two mice. The gene is then turned off in both mice and drug administered to one mouse but not the other. Measurements are then taken over time for both mice to determine the rate at which harmful protein is cleared from the system of each mouse. The drug is determined efficacious if it increases the rate at which the harmful protein is cleared from the system as compared to the rate it is cleared from the system of the mouse with no drug.

EXAMPLES The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

EXAMPLE 1 COPPER DEPRIVATION OF NEONATAL Prnp^0/0 MICE Since copper increases rapidly in the neonatal mouse CNS, a protocol was developed to deprive mice of copper at a very early stage of development. Accordingly, female mice with four day old pups were fed a copper deficient diet to in turn deprive the developing pups of copper. Mice with an ablated endogenous PrP gene (Prnp⁰⁰), mice of the parent strain FVB, and mice expressing higher copy number of PrP (Tg(MoPrP-A)4053/FVB) were all examined for neonatal sensitivity to copper in their diet. Tg(MoPφ-A) 4053/FVB were obtained by microinjection of one cell zygotes collected from FVB mice (Telling et al., 1996). The transgenic line was maintained by breeding the transgene-positive founder with wild-type FVB animals (Charles River, Wilmington -Ma). The PrP deficient animals used were obtained by backcrossing hemizygous Prnp^{+ 0} animals (Bϋeler et al., 1992, Prusiner et al., 1993b) with FVB animals for 10 generations before interbreeding to homozygosity. Breeding and screening of transgenic (Tg) mice were performed as previously described (Prusiner et al, 1993b; Scott et al., 1989).

Wild type and transgenic mice were mated to establish a breeding colony. Four days after parturition, the mouse dams with pups were divided into two dietary treatments, -Cu and +Cu . The-Cu group was fed a copper depleted diet (TD 80388; Teklad, San Diego, CA) and drank purified deionized water (Millipore, Bedford, MA). This diet contained less than 0.45 mg of copper per kilogram of pellets. Mice in the +Cu group were fed a diet containing 19.4 mg/kg of copper, and drank normal drinking water ad libitum. Mice were housed in polycarbonate disposable cages with stainless steel frame and a cardboard next for pups. The mice were weaned at 4 weeks of age, their genotype determined, and placed five each in disposable polycarbonate cages with elevated stainless steel frame. The mice were checked for clinical symptoms, weighed daily and continued to receive the assigned diet for an additional two weeks. The experiment was terminated after 42 days. The animals were euthanized in terminal stages of disease or after 42 days. Mice euthanized in Halothan or CO₂ were decapitated and trunk bleed into 1.5 ml Eppendorf tubes. Blood was kept at room temp for 30 minutes, clots removed, and spun at 1000 φra for 10 minutes. Collected serum was stored at -20 °C for the ceruplasmin assay and copper measurements. Brain, liver, and kidneys were removed, immediately frozen, and stored at -70 °C.

Cerulopasmin activity in serum was used to monitor the effectiveness of the copper deprivation scheme. Similar levels of ceruloplasmin were detected in Prnp^0/0, FVB and Tg(MoPrP-A)4053/FVB mice fed a normal diet and sacrified at 46 days of age (Figure 1). In contrast, ceruloplasmin in Prnp⁰⁰ mice measured at the time of sacrifice was 16% of that found in control Prnp^0/0 mice. The ceruloplasmin in FVB and Tg(MoPrP-A)4053/FVB mice at the time of sacrifice was 16% and 13% of that found in the control mice, respectively. These measurements demonstrate that all three lines of mice responded similarly to dietary copper restriction as measured by ceruloplasmin activity. Copper levels in serum and brain were also measured in control and experimental mice as shown in Table 2 below.

Tissue Diet p_mpo/o FVB Tg(MoPφ-A) Cu [μg/g] Cu [μg/g] Cu [μg/g] brain +Cu 1.77 ± 0.08 2.40 ± 0.20 2.22 ± 0.07 -Cu 0.30 ± 0.02 0.27 ±0.04 0.17 ± 0.00 serum +Cu 0.46 ± 0.30 0.47 ± 0.03 0.31 ± 0.02 -Cu 0.001 ± 0.003 0.008 ± 0.009 0.004 ± 0.007 liver +Cu 4.08 ± 0.72 4.64 ± 0.27 3.44 ± 0.25 -Cu 1.87 ± 0.49 1.13 ± 0.15 1.62 ± 0.48 kidney +Cu 2.34 ± 0.19 4.35 ± 0.32 2.20 ± 0.32 -Cu 1.47 ± 0.09 1.73 ± 0.30 0.63 ± 0.18

Serum copper was levels were similar in Prnp^0/0 and FVB mice fed normal diet while overexpression of PrP^c in Tg(MoPrP-A)4053/FVB mice lowered the serum copper level approximately 30%. Serum copper levels in all three lines were virtually undetectable when the mice were sacrificed at 42 days after dietary copper deprivation initiation. Copper levels in the brains of Prnp^{0 0} mice were only slightly lower than those of FVB and Tg(MoPrP- A)4053/FVB mice when fed a normal diet. This was suφrising since copper levels in brain microsome preparations from Prnp^0/0 mice were reported to be approximately 20% of those found in controls (Brown et al., 1997a). Brain copper levels in all three lines fed a copper restricted diet were about 10% of those found in controls when the mice were sacrificed at 42 days after dietary copper deprivation was initiated. In liver and kidney, all the mice showed substantially reduced copper levels after dietary copper deprivation except for the Prnp^0/0 mice. Although kidney copper levels in p_mp^θ/o _mj_{ce were} approximately 50% of those in FVB mice fed a normal diet, there was only a modest reduction in kidney copper when the Prnp⁰⁰ were fed a copper restricited diet. Curiously the kidney copper levels in the Tg(MoPrP-A)4053/FVB mice fed a normal diet were similar to those in Prnp⁰⁰ mice. In contrast to Prnp⁰⁰ mice, kidney copper in

Tg(MoPrP-A)4053/FVB mice after dietary copper deprivation was substantially reduced.

Serum ceruloplasmin was determined spectrophotometricly by oxidation of p- phenylenediamine as described (Rice et al, Anal. Biochem. 3:452-456 (1962 ). Briefly, 20 μl or sera was mixed with 200 μl of 0.1% (w/v) p-phenylenediamine (PPD) solution in 1.2 M acetate buffer, containing 40 μM of EDT A. The mixture was incubated for 15 min at 37°C and reaction stopped by 1 ml of 1% (w/v) NaN₃. The absorbance was read at 540 nm and the ceruloplasmin activity was calculated from a standard calibration curve.

All glassware was washed, soaked in 30% nitric acid overnight and rinsed extensively in double-distilled water. The brain, kidneys and liver were harvested, rinsed in distilled water to remove excess blood, weighed, and stored at -70 °C. Tissues were digested in 5 ml nitric acid with 5 drops of sulphuric acid (Intsra grade, J.T. Baker, Philipsburg, NJ) at room temp for 3-4 hours until all digested. A blank control with no organ was used with every set of digestions. The digested organs were boiled in presence of acid washed boiling beads until nearly all the nitric acid was boiled off. An additional 2 drops of nitric acid were added and boiled off, and the tissue allowed to char to black. Next, a 2 ml aliquot of hydrogen peroxide (Sigma, St. Louis, MO) was added and allowed to boil off, followed by 1 ml aliquots at a time until liquid clarified or became very pale yellow. After all the H₂0₂ was boiled off, the remaining residue was dissolved in 2.5 ml 20% nitric acid.

Mice starved for copper exhibited diminished liver weights at autopsy. The livers of Prnp^0/0 mice on a copper restricted diet were 64% as large as those of control Prnp^0/0 mice fed a normal diet as shown in Table 3 below. Diet _Pmpo/o FVB Tg(MoPrP-A) wt [g] wt [g] wt [g] body +Cu 21.24 ± 0.55 20.18 ± 0.37 21.76 ± 0.66

-Cu 15.28 ± 0.21 13.54 ± 1.27 11.70 ± 1.02 brain +Cu 0.47 ± 0.01 0.42 ± 0.02 0.46 ± 1.02

-Cu 0.43 ± 0.02 0.37 ± 0.01 0.39 ± 0.00 liver +Cu 1.28 ± 0.10 0.73 ± 0.03 1.19 ± 0.15

-Cu 0.83 ± 0.04 0.52 ± 0.08 0.57 ± 0.07 kidney +Cu 0.17 ± 0.02 0.12 ± 0.01 0.14 ± 0.01

-Cu 0.15 ± 0.01 0.14 ± 0.01 0.12 ± 0.01

Similarly, the livers of FV13 and Tg(MoPrP-A)4053/FVB mice on a copper restiricted diet were 71% and 48% as large at the time of sacrifice as those of control mice fed a normal diet, respectively. By contrast, no significant decrease in brain or kidney weights were found in mice fed a copper restricted diet. Copper measurements were performed in a 2380 Perkin Elmer flameless graphite furnace atomic absoφtion spectrophotometer at a wavelength of 324.7 nrn after rapid atomization at 2300*C in a graphite tube cuvette HGA-70 (Perkin Elmer). The absolute amount of copper was calculated from standard calibration curve after subtracting blanks. The copper concentration for each organ are expressed in μg/g of wet tissue weight.

After a series of studies with weanling mice in which we were unable to achieve severe copper deprivation as noted above, we implemented a protocol using neonates. At four days of age, mothers and offspring were placed on a copper deficient diet. The three different lines of mice Prnp^0/0, FVB, and Tg(MoPrP-A)4053/FVB described above were employed in these studies. After 42 days, the experiment was terminated because so many of copper-deprived animals that remained alive appeared very ill. Mice sacrificed on the terminal day of the experiment were 46 days of age.

After four days on the copper deficient diet, the Prnp⁰⁰ neonates began to exhibit signs of CNS dysfunction. By 30 days on the copper deficient diet, 50% of the Prnp⁰⁰ mice displayed ataxia, tremor and paresis (Figure 2). These copper deprived Prnp^0/0 mice exhibited a yellow tint in their fur, had a scuffy appearance due to their failure to groom themselves and appeared emaciated. Compared to Prnp^0/0 mice fed a normal diet, the copper deficient Prnp^0/0 mice had substantially lower body weights. At the time of death, all mice deprived of dietary Cu2+ exhibited widespread astrocytic gliosis in the brainstem and cerebellum with few pathologic changes seen in the cerebrum. In contrast, none to the control mice fed a normal diet and sacrificed at 46 days of age showed any neuropathological changes. In the mice fed a copper restricted diet, the main neuropathological feature was reactive astrocytic gliosis without obvious nerve cell loss, nerve cell degeneration or neuronal dystrophy. Reactive astrocytic gliosis was most obvious in all copper deficient animals in the cerebellar cortex and white matter. The degree of reactive astrocytic gliosis in each animal was estimated by a semiquantitative method which assesses the area of the cerebellar cortex occupied by reactive astrocytes and their processes as revealed by GFAP immunohistochemistry (Figure 3). The control group of five brains consisted of one Prnp⁰⁰, two FVB mice and two Tg(MoPrPA)4053/FVB mice all fed a normal diet. The copper deprived mice, all of which had signs of neurologic dysfunction, consisted of four Prnp^0/0 mice, eight FVB mice and 16 Tg(MoPrP-A)4053/FVB mice. Each group of copper deficient mice showed statistically significant differences in the proportion of cerebellar cortex exhibiting GFAP immunoreactivity. Student's t test probabilities comparing gliosis in copper deprived mice to that in the control group were: P=0.011 for Prnp⁰⁰ mice, P=0.0072 for FVB mice and P=0.0001 for Tg(MoPrP-A)4053/FVB mice. About 50% of the copper deficient Tg(MoPrP-A)4053/FVB mice also showed varying degrees of reactive astrocytic gliosis in the outer layers of the cerebral cortex, the cingulate gyrus, caudate nucleus and thalamus.

EXAMPLE 2 COPPER DEPRIVATION OF ADULT Prnp⁰⁰ MICE Mice with an ablated endogenous PrP gene, mice of the parent strain FVB, and mice expressing higher copy number of PrP were all examined for sensitivity to copper in their diet.

To this end, Prnp^0/0, FVB, and Tg(MoPrP-A)4053/FVB mice were maintained on a normal, copper-sufficient diet, or fed a copper deficient diet (TD 80388) and examined for potential phenotypes. The strains of mice used were produced as per Example 1. Greater than 90% of Prnp^0/0, FVB, and Tg(MoPrP-A)4053/FVB mice maintained on a normal, copper-sufficient diet developed normally and remained well for more than 300 days. The Prnp^0/0 mice were observed for more than 760 days at which time less than 10% had developed. Lifespan studies of FVB and Tg(MoPrP-A)4053/FVB mice at 300 days are consistent with these results. From these findings, we conclude that genetic ablation of the PrP gene in mice, absent copper deprivation, is not deleterious.

Prnp^0/0 Mice fed a copper deficient diet showed a shaφly declining survival rate, with less than half of the animals surviving beyond 30 days from initiation of copper deprivation. In contrast to the Prnp^0/0 mice, only 5% of the FVB and Tg(MoPrP-A)4053/FVB mice fed a copper deficient diet for 30 days displayed signs of neurologic dysfunction (Figure 2). Moreover, sfter 42 days on a copper deficient diet, 28% of the FVB mice and 27% of the Tg(MoPrP-A)4053/FVB mice had been sacrificed after becoming severely emaciated compared to 68% of the Prnp^0/0 mice.

While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

WHAT IS CLAIMED IS:

1. A transgenic non-human animal having a genome comprised of an endogenous PrP gene altered to decrease PrP expression; wherein the transgenic animal is characterized by a pathological phenotype upon copper deprivation associated with a decrease in endogenous PrP expression, and wherein copper is administered to the transgenic animal in an amount sufficient to suppress the pathological phenotype.

2. The transgenic animal of claim 1, wherein both alleles of the endogenous PrP gene is ablated; wherein the transgenic animal is a mammal selected from the group consisting of mouse, hamster, rat, rabbit, guinea pig, cow, sheep, deer, goat, pig, chicken, turkey, dog and cat; and wherein the copper is administered orally.

3. A transgenic animal produced by the process comprising the steps of: ablating both alleles of an endogenous PrP gene of an animal; and administering copper to the animal; wherein the administration of copper suppresses a pathological phenotype associated with the ablation of the endogenous PrP gene.

4. A transgenic animal having a genome comprised of: an endogenous PrP gene altered to decrease PrP expression; and an exogenous PrP gene; wherein the transgenic animal is characterized by a pathological phenotype upon copper deprivation associated with a decrease in endogenous PrP expression, and wherein copper is administered to the transgenic animal in an amount sufficient to suppress the pathological phenotype.

5. A transgenic animal having a genome comprised of: an endogenous PrP gene altered to decrease PrP expression; and an exogenous PrP gene from a genetically diverse species; wherein pathological phenotypes upon copper deprivation associated with a decrease in endogenous PrP expression are suppressed by the expression of the exogenous PrP gene.

6. The transgenic animal of claim 5, wherein the transgenic animal is a mouse, and the exogenous PrP gene is from a genetically diverse species selected form the group of: human, cow and sheep.

7. The transgenic animal of claim 5 wherein the exogenous PrP gene is an artificial gene comprised in part of codons from a PrP gene of a genetically diverse species, the transgenic animal being characterized by being susceptible to infection with a prion which generally only infects a genetically diverse animal.

8. The transgenic animal of claim 5, wherein: the transgenic animal is selected from the group consisting of a mouse, hamster, rat, rabbit, and guinea pig; and the exogenous gene is from a genetically diverse animal selected from the group consisting of a human, cow, sheep, goat, pig, chicken, turkey, dog and cat.

9. The transgenic animal of claim 5, wherein: the transgenic animal is selected from the group consisting of cow, sheep, goat, pig, chicken, turkey, dog and cat; and the exogenous gene is from a genetically diverse animal selected from the group consisting of a human, a mouse, hamster, rat, rabbit, and guinea pig.

10. The transgenic animal of claim 5, wherein the transgenic animal is a mouse and the exogenous PrP gene is from a human.

11. The transgenic animal of claim 5, wherein: the exogenous PrP gene is from a genetically similar animal; and wherein pathological phenotypes associated with a decrease in endogenous PrP expression are suppressed by the expression of the exogenous PrP gene.

12. The transgenic animal of claim 11, wherein: the transgenic animal is selected from the group consisting of a mouse, hamster, rat, rabbit, and guinea pig; and the exogenous gene is from a genetically similar animal selected from the group consisting of a mouse, hamster, rat, rabbit, and guinea pig.

13. The transgenic animal of claim 5, wherein the exogenous PrP gene is operably linked to an inducer sequence which effects expression of the PrP gene.

14. The transgenic animal of claim 13, wherein the inducer sequence is activated by administration of a compound to the transgenic animal, thereby affecting expression of the exogenous PrP gene.

15. The transgenic animal of claim 14, wherein the inducer sequence comprises an inducible transactivator sequence and an operator sequence wherein both are operably linked to the exogenous PrP gene in a manner so as to control expression of the exogenous PrP gene.

16. The transgenic animal of claim 15, wherein the inducible transactivator sequence is a tetracycline transactivator (tTA) sequence and the operator sequence a heptamer of a tetracycline operator sequence (tetO).

17. A transgenic animal produced by the process comprising the steps of: ablating the endogenous PrP gene of a host animal; operatively incoφorating an exogenous PrP gene into the genome of the host animal wherein the exogenous PrP gene is comprised of a PrP gene from a genetically diverse species; wherein the transgenic animal is susceptible to infection with a prion which generally only infects a genetically diverse test animal; wherein the transgenic animal is further characterized by a pathological phenotype upon copper deprivation associated with a decrease in endogenous PrP expression; and wherein copper is administered to the transgenic animal in an amount sufficient to suppress the pathological phenotype.

18. The animal of claim 17, wherein the exogenous PrP gene is operatively incoφorated in the genome along with a promoter which enhances expression of the exogenous PrP gene.

19. A transgenic animal produced by the process comprising the steps of: ablating the endogenous PrP gene of a host animal; operatively incoφorating an exogenous PrP gene into the genome of the host animal wherein the exogenous PrP gene is comprised of a PrP gene native to the host animal with one or more of its codons replaced with a corresponding codon of a PrP gene of a genetically diverse animal and wherein one or more of the replacing codons of the genetically diverse test animal is a codon indicative of a prion disease in the genetically diverse animal; wherein the transgenic animal is susceptible to infection with a prion which generally only infects a genetically diverse test animal; wherein the transgenic animal is further characterized by pathological phenotypes upon copper deprivation associated with a decrease in endogenous PrP expression; and wherein copper is administered to the transgenic animal in an amount sufficient to suppress the pathological phenotypes.

20. The animal of claim 19, wherein the exogenous PrP gene is operatively incoφorated in the genome along with a promoter which enhances expression of the exogenous PrP gene.

21. The animal of claim 19, wherein the host animal is a mouse and the genetically diverse animal is selected from a human, a cow and a sheep.

22. A method of detecting prions in a material, comprising the steps of: providing a PrP transgenic animal as claimed in claim 4; administering to the transgenic animal sufficient copper to control the pathological phenotype observed upon copper deprivation; inoculating the transgenic animal with a material suspected of containing prions; and observing the transgenic animal for a symptom of prion disease.

23. The method of claim 22, wherein the transgenic animal is a mouse and the mouse is observed for ataxia.

24. A method of determining the efficacy of a drug, comprising: providing a PrP transgenic animal as claimed in claim 4; administering to the transgenic animal sufficient copper to control the pathological phenotype observed upon copper deprivation; administering a drug to the transgenic animal; inoculating the transgenic animal with a material suspected of containing prions; and observing the transgenic animal for a symptom of prion disease.

25. The method of claim 24, wherein the transgenic animal is a mouse.